As a method for the qualitative or quantitative determination of a trace amount of a substance contained in an in vivo sample such as blood or urine, immunochemical assay methods are generally used due to their high sensitivity. Of these methods, a so-called immunochromatographic method using chromatography is widely used in many fields, e.g., for clinical examination in a hospital and an assay test in a laboratory.
As a method of detecting an analyte by an immunochromatographic method, a variously labeled specific binding substance (antibody) is reacted with an analyte to be detected (antigen) on a chromatographic material to form a complex of the analyte and the labeled specific binding substance (antigen-antibody complex), and this complex is found (detected) by a variety of means. The label includes radioisotope, chromophore, fluorophore, enzyme, etc. The detecting means include a radiation detector, a spectrophoto meter etc. and visual detection.
JP-A-64-32169 discloses a qualitative assay method by immunochromatography using a colloidal-particles-labeled specific binding substance (antibody) which is chromatographically mobile and capable of generating visually detectable signal, and an apparatus therefor. This publication discloses one means which allows visual detection.
The method described in the above publication is a method for assaying the presence or absence of an analyte (antigen) in a sample or its amount, and comprises (a) bringing a sample containing an analyte (antigen) into contact with a chromatographic medium, (b) moving the above colloidal-particles-labeled substance (labeled antibody) on the chromatographic medium, to allow at least part of the colloidal-particle-labeled substance (labeled antibody) to move to a reactive site and cause a binding reaction, and (c) determining detectable response caused by the colloidal substance in the reaction site for denoting the presence or absence of the analyte (antigen) in the sample and its amount.
Japanese PCT Laid-open Publication No. 1-503174 discloses an apparatus provided with a labeled first antibody which is to specifically bind to an analyte to be detected (antigen) (to be referred to as "labeled first antibody" hereinafter),and is freely mobile on a chromatographic medium in a wet state, and an unlabeled second antibody which is to specifically bind to the analyte (antigen) (the second antibody having an antigen binding portion different from that of the first antibody) (to be referred to as "unlabeled second antibody" hereinafter), and fixed on the chromatographic medium, the apparatus being capable of detecting the presence of the analyte, in a detection area on the chromatographic medium by adding a liquid sample containing the analyte to one end of the chromatographic medium so that the liquid sample moves through the chromatographic medium, reacts with the labeled first antibody and then reacts with the unlabeled second antibody. The principle used in the above apparatus is a so-called (immunochemical) sandwich method.
For determining the amount of an analyte in a sample, conventionally, there is employed a method in which a sample containing an analyte is properly diluted and subjected to a qualitative reaction with a measuring reagent having a predetermined sensitivity and a maximum dilution ratio at which positivity is shown is multiplied by the sensitivity to determine a semi-quantitative assay value, or qualitative reactions are carried out with reagents having different sensitivity without diluting an analyte and the sensitivity of the reagent at which the analyte shows positivity is taken as a semi-quantitative assay value. The method of quantitative analysis includes a liquid-phase assay carried out in a container such as a microtiter well and a solid-phase assay carried out on a chromatographic medium. In the methods described in the above two publications, samples are diluted for carrying out the quantitative determination.
JP-A-4-351962, laid-open recently, discloses a specific-binding analysis method which permits semi-quantitative assay without diluting a sample and an apparatus therefor. In the method described in this publication, for the qualitative or quantitative determination of an analyte in a sample by a chromatographic method, a specific substance is allowed to be present in a measurement system and the amount of a labeled substance to be measured as an index for the analyte is decreased due to the presence of the specific substance, so that there can be consequently obtained a result similar to the result obtained by diluting a sample containing the analyte (to be referred to as "dilution effect" hereinafter).
In a typical method described in the above publication, an analyte (a) added to a sample addition portion contacts a predetermined amount (concentration) of a specific substance (b), which is present on a chromatographic material without being fixed, and a predetermined amount of a labeled specific-binding substance (e), which is present on the chromatographic material without being fixed, (in a site where the specific-binding substance is present). A certain amount of the analyte (a) bonds to the specific substance (b). When, however, an excess of the analyte (a) over the specific substance (b) is present, the excess of the analyte (a) moves to a portion (detection portion) where a specific-binding substance (specific substance (b) or a substance (g) capable of binding to that portion of the analyte (a) which the specific substance (b) bonds to) is present being fixed to the chromatographic material while the excess of the analyte (a) retains a portion capable of binding to the specific substance (b). The analyte (a) which has no portion capable of binding to the specific substance (b), or at least has bound to the specific substance (b), passes the detection portion. Only a complex (f) of the analyte (a)--the labeled specific-binding substance (e), which at least has a portion capable of binding to the specific substance (b), is fixed on the detection portion, and this fixed complex (f) is detected by various means.
It has been generally required to dilute a sample for semi-quantitative assaying an analyte in the sample. Further, the method using reagents having different sensitivity, which does not require the dilution of a sample, has a defect in that the determination is difficult since the intensity of one reaction in one measurement sensitivity is different from the intensity of another reaction in another measurement sensitivity, so that this method has been scarcely practically used.
In the field of medical treatment, clinical examination in particular, the operation of diluting a sample increases the possibility of an examiner being infected with pathogen from blood, urine, etc., as testing samples. Further, when a large number of samples are semi-quantitative assayed, the operation efficiency can be remarkably improved if the dilution operation is not necessary. It is therefore an object of the present invention to provide a simple immunochemical semi-quantitative assay method which does not require the dilution of a sample, and an apparatus therefor.
Like the present invention, the above-described JP-A-4-351962 discloses an invention which does not require the dilution of a sample. In the method of this publication, many factors are concerned with the adjustment of "dilution effect" of a sample, and it is difficult to adjust the dilution effect, so that no narrow semi-quantitative assay value width can be set and that the method has a problem in measurement accuracy. It is further an object of the present invention to provide an immunochemical semi-quantitative assay method in which the number of factors concerned with the adjustment of "dilution effect" is small so that the dilution effect can be easily adjusted and the sensitivity can be improved, i.e., a semi-quantitative assay value width can be narrowly set, and an apparatus therefor.